Picornaviruses are single positive strand RNA viruses. The genome of approximately 7,500–8,250 bases contains a single long open reading frame encoding a large polyprotein, usually approximately 230 kDa. The polyprotein is processed auto-proteolytically by virus-encoded proteases. The ‘primary’ cleavages separate the polyprotein into the structural capsid protein (P1), and non-structural proteins involved in virus replication (P2 and P3), see FIG. 1. All three primary proteins (P1, P2 and P3) undergo post-translational cleavage to produce mature proteins (reviewed in reference [1]).
In the case of the rhino- and enteroviruses this processing is achieved by the 2A proteinase cleaving at its own N-terminus, whilst in the aphtho- and cardioviruses the 2A protein mediates cleavage at its own C-terminus[1]. In the latter viruses this cleavage is mediated by an oligopeptidic tract (18 amino acids) corresponding to the complete 2A protein of Foot-and-Mouth Disease Virus (FMDV), or, just the C-terminal region of the larger (˜145 amino acids) cardiovirus 2A protein[2]. The sequence is able to mediate cleavage in entirely foreign protein contexts[3] and has been used in a wide range of co-expression studies[4-6]. The corresponding primary cleavage in the hepato- and parechoviruses is mediated by the 3C proteinase (see FIG. 1).
It is known that large numbers of membrane vesicles are produced in the cytoplasm of picornavirus-infected cells[7]. These vesicles are thought to arise from the transitional smooth areas of the Endoplasmic Reticulum (ER) that produce transport vesicles which move proteins from the ER to the Golgi[8]. It is also known that Brefeldin-A, a drug that blocks the formation of ER-derived transport vesicles, is one of the most potent known inhibitors of picornavirus replication[9,10] and that normal membrane transport from the ER to the Golgi is blocked in picornavirus infected cells[11,12]. Picornavirus infection appears, therefore to change the balance of membrane budding and fusion which is normally maintained between the ER and Golgi compartments. Transport vesicles leaving the ER do not appear to fuse with the Golgi, but are diverted from the secretory pathway to function at sites of virus replication, explaining why the Golgi is absent later in infection. Indeed, the loss of the Golgi is coincident with the formation of virus replication complexes in the cytoplasm. These appear as rosettes of large vesicles surrounding a compact central membrane system.
As early as 1982 Semler and co-workers demonstrated the association of the non-structural protein 3AB with membranes[13]. Other non-structural viral proteins 3AB, 2BC, 2B, 2C have since been shown to localise to crude membrane fractions[14], and to the ER[15]. The membrane association of 3A and 3AB has been explained by the presence of 20 hydrophobic amino acids at the C-terminus of 3A, and deletion of this region abrogates membrane binding. The membrane association of 2C and 2BC was proposed via an amphipathic helix that produces a hydrophobic patch at the N-terminus of 2C[12].
Importantly, protein 2C has been localised to electron-dense coats within the regions of ER membrane producing cytoplasmic vesicles, and within membrane vesicles of the replication complex[14]. The observation that 3A, 3AB, 2BC and 2B bind membranes makes them strong candidates as inhibitors of membrane traffic in cells. Indeed recent studies show that 2BC and 2C induce the formation of vesicles[14], and 3A and 2B block secretion, when expressed alone in cells[11, 12].
In picornaviruses, RNA replication is solely a function of the P2 and P3 regions of the polyprotein. A full-length negative sense copy of the genomic, positive sense, RNA is synthesised and acts as a template for the synthesis of full length positive RNA strands. In the case of the entero- and rhinoviruses 2A is a proteinase[1] and forms part of the P2 primary cleavage product. The function of protein 2B is unknown, protein 2C is discussed in detail below, protein 3A is thought to function as a ‘donor’ for the protein 3B (or VPg) which becomes covalently bound to the 5′ terminus of both positive and negative strand RNA. Protein 3C is a proteinase (see reference [1]), which serves to mediate a primary cleavage between P2 and P3 and subsequently processes P2 and P3 (‘secondary’ cleavages).
Protein 2C is the second largest mature protein (approximately 37 kDa) and is the most highly conserved picornavirus protein. Indeed, homologues of 2C are found within other members of the picornavirus ‘supergroup’—comprising both (non-picornavirus) animal viruses (caliciviruses, hepatitis E viruses; various insect viruses) and numerous plant viruses (comoviruses, nepoviruses).
Based on sequence alignments it was proposed that protein 2C contained NTP binding motifs[16] and could function as a helicase. Although it has not proved possible to demonstrate helicase activity this notion has been stated in the literature and is believed to be correct within the current understanding of picornavirus protein function[41-44].
Protein 2C is known to have ATPase/GTPase activity[17,18]. Membrane binding activity maps to the N-terminal region (amino acids 21–54) of poliovirus 2C, containing one of the putative amphipathic helices (See FIG. 2, panel A). Protein 2C is not glycoslyated nor cleaved by signal peptidases[19]. Expression of 2C induces membrane proliferation and blocks the exocytic pathway[20,21].
Furthermore, 2C is an RNA-binding protein with sites mapped to the N- and C-terminal regions (amino acids 21–45 and 312–319)[19], summarised in FIG. 2, panel A. The RNA sequences bound by 2C were determined to be a stem of a clover-leaf structure SEQ ID NO:7 (see FIG. 2, panel B) initially characterised in the positive RNA sense and predicted to be present in the negative sense (anti-genome) RNA. Interestingly, one can see that one component of the RNA-binding domain is contained within that determined for membrane binding.
The specificity of signalling molecules is greatly enhanced by their sub-cellular compartmentalisation within the cell. The distribution of these molecules is governed by their association with (membrane-bound) anchoring proteins which place these enzymes close to their appropriate effectors (reviewed in [20–22]). The dual-specificity kinase anchoring protein 2 (D-AKAP2) binds the regulatory subunits (both RI and RII) of protein kinase A (PKA). Originally identified by their co-purification with RII after cAMP-sepharose chromatography[23], AKAPs were further characterised using their property of binding RII immobilised to nitrocellulose or similar solid-phase supports[24]. D-AKAP2 sequences are available for mouse[25], human[26] and we have completely sequenced the porcine D-AKAP2[27] identified by the yeast screen. In each case a putative ‘regulator of G-protein signalling’ (RGS) domain is present, although sequence similarities between D-AKAP2 and the RGS domains of other proteins is low. The binding of PKA regulatory subunits is thought to be mediated by an amphipathic helix[21]. Inspection of aligned D-AKAP2 sequences shows similarity between the C-terminus and RII binding sites determined for other AKAPs[21]. The PKA (RII)/D-AKAP2 binding is high (low nanomolar) affinity[28] but does require prior dimerisation of the RII subunit[19]. D-AKAP2 shows a wide tissue distribution although its location(s) at the sub-cellular level has not been determined.
It has now be found that a strong interaction exists between FMDV protein 2C and the cellular membrane protein D-AKAP2. When FMDV 2C is used as ‘bait’ in the 2-hybrid yeast screen, a strong interaction is observed with the C-terminal region (50 amino acids) of D-AKAP2. The results from our genetic screen have been confirmed by direct biochemical assays. D-AKAP2 binds protein kinase A (PKA) and has dual specificity for both the regulatory subunits (RI and RII) of this enzyme (see above). The yeast two-hybrid 2A:D-AKAP2 interaction was confirmed by direct biochemical binding assays using FMDV 2C translated in vitro and from infected-cell lysates. We extended these observations by the analysis of a site-directed mutagenetic form of 2C. A single mutation in the NTP-binding motif (K116 to Q) renders this form of 2C unable to reorganise the membrane structure of transfected cells but does not alter the binding to D-AKAP2 in biochemical assays.
Since the membrane binding region of 2C has been mapped to residues 21 to 54, we assume this is the site of interaction with D-AKAP2.
Whilst the present invention is founded upon observations made with FMDV 2C, the strong homology between the 2C proteins of all picornavirus, caliciviruses, comoviruses, nepoviruses etc. is recognised in the art and acknowledged in the literature.
The reference to “picornavirus protein 2C” herein refers not only to the 2C protein of picornaviruses alone, but also to the 2C protein of the picornavirus “supergroup” which includes calciviruses, hepatitis E viruses, comoviruses and nepoviruses.